Embryos in the morula or blastocyst stage can be frozen in a cryoprotectant solution and later thawed. The thawed embryos have a reasonably good viability rate and are transferred to the uterus of recipient animals for further gestation. The typical procedure in the embryo transfer industry requires a serial rehydration of embryos following thawing. The embryo must be removed from the freezing container, and handled by a technician. The process can take at least 30 to 40 minutes. The complicated transfer methods devised by prior investigators in this field require the concentration of cryoprotectant in the embryo freezing solution to be decreased slowly after thawing to avoid lysing embryonic cell membranes. See e.g. Miyamoto, H. and Ishibashi, T., "The Protective Action of Glycols Against Freezing Damage of Mouse and Rat Embryos", J. Reprod. Fert, 54: 427-32 (1978). This "step-wise rehydration" of the embryo has been assumed to be a necessary part of embryo handling. More recently, some investigators have rehydrated thawed embryo cells using sucrose as an osmotic buffer. Renard, J., Ozil, J., and Heyman, Y., "Cervical Transfer of Deep Frozen Cattle Embryos", Theriogenology, 15: 311-320 (1981).
In an embryo transfer technique, the primary means for cryopreservation of mammalian embryos employs the use of permeating cryoprotective agents. Glycerol is the most commonly used cryoprotectant today. Under the "step-wise rehydration" technique, embryos are most often frozen in semen straws, and upon thawing, are removed from the straws through a series of solutions containing decreasing concentrations of cryoprotectant and then reloaded into a semen straw for nonsurgical transfer to a recipient female. All embryo handling procedures require the use of a microscope, sterile supplies and solutions, and must be performed by a skilled technician trained in these procedures. Therefore, developing a process by which the frozen embryo can be thawed within the straw and transferred directly to a recipient female without seriously diminishing pregnancy rates would be very important commercially.
Currently, the most commonly employed cryoprotective agent is glycerol. Leibo, S. P. "A One-Step Method for Direct Nonsurgical Transfer of Frozen-Thawed Bovine Embryos", Theriogenology, 21: 767-90 (1984). Among the other cryoprotectants investigators have looked at using is ethylene glycol, which has been researched as a cryoprotectant for mammalian embryos, but not using a direct transfer method. Heyman, Y., Vincent, C., Garnier, V., and Cognie, Y., "Transfer of Frozen-Thawed Embryos in Sheep", Veterinary Record, 120: 83-85 (1987). The procedure published by Y. Heyman, et al, in 1987, using ethylene glycol as a cryoprotectant for sheep embryos, requires a "one-step" dilution or rehydration with sucrose. Other investigators found ethylene glycol to be a good cryoprotectant for murine embryos, but their method still requires a three-step rehydration with a modified Krebs-Ringer bicarbonate medium. Miyamoto, H. and Ishibashi, T. "The Protective Action of Glycols Against Freezing Damage of Mouse and Rat Embryos", J. Reprod. Fert., 54: 427-32 (1978). Miyamoto, H. and Ishibashi, T., "Survival of Frozen-Thawed Mouse and Rat Embryos in the Presence of Ethylene Glycol", J. Reprod. Fert., 50: 373-75 (1977). Other investigators have looked at direct transfer methods, but these experiments have required a combination of glycerol and sucrose as the cryoprotective agent. Massip, A., Van Der Zwalmen, P. and Ectors, F., "Recent Progress in Cryopreservation of Cattle Embryos", Theriogenology, 27: 69-79 (1987).
One rehydration technique referred to as the "one-step" method has one rehydration step rather than a multiple step-wise rehydration. Leibo, S. P. "Embryo Transfer Method and Apparatus", U.S. Pat. No. 4,380,997, issued Apr. 26, 1983. However, even the "one-step" method involves special handling of embryos in the field because it still requires dilution of the cryoprotectant upon thawing. Although patented and described in the scientific literature, the results of this procedure "have been less than satisfactory in that only a 26% pregnancy [rate] has been achieved overall." Leibo, S. P. "A One-Step Method for Direct Nonsurgical Transfer of Frozen-Thawed Bovine Embryos", Theriogenology, 21: 767-90 (1984). Leibo reported highly variable results for individual sets of frozen-thawed embryos with pregnancy rates ranging from 0% and 6% to 55% and 63%. It is believed that the technician performing the thaw exerts a significant effect on pregnancy rates suggesting that this procedure is too elaborate to be reproducible enough to meet commercial needs.